Sealing of bottles: Complete filling of each bottle by adding enough dilution water that insertion of the stopper leaves no bubbles in the bottle. Mix the sample by turning the bottle manually several times unless a DO probe having a stirrer is used immediately to measure initial DO concentration. As a precau- tion against drawing air into the dilution bottle during incuba- tion, use a water seal. Obtain satisfactory water seals by invert ing bottles in water bath or by adding water to the flared mouth of special BOD bottles. Place a paper or plastic cup or foil cap over flared mouth of bottle to reduce evaporation of the water seal during incubation.